U.S. patent application number 17/265882 was filed with the patent office on 2021-06-03 for method and apparatus for providing an isolated single cell.
The applicant listed for this patent is Edmond WALSH. Invention is credited to Edmond WALSH.
Application Number | 20210162401 17/265882 |
Document ID | / |
Family ID | 1000005403134 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210162401 |
Kind Code |
A1 |
WALSH; Edmond |
June 3, 2021 |
METHOD AND APPARATUS FOR PROVIDING AN ISOLATED SINGLE CELL
Abstract
Methods and apparatus for providing an isolated single cell are
provided. In one disclosed arrangement, a test body of liquid is
formed on a substrate surface. A contact angle between the test
body of liquid and the substrate surface is lower than an
equilibrium contact angle. An optical image of the test body of
liquid is analysed to determine whether one and only one cell is
present in the test body of liquid.
Inventors: |
WALSH; Edmond; (Oxford,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WALSH; Edmond |
Oxford |
|
GB |
|
|
Family ID: |
1000005403134 |
Appl. No.: |
17/265882 |
Filed: |
August 8, 2019 |
PCT Filed: |
August 8, 2019 |
PCT NO: |
PCT/GB2019/052233 |
371 Date: |
February 4, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/5088 20130101;
B01L 2300/025 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 10, 2018 |
GB |
1813094.8 |
Claims
1. A method of providing an isolated single cell, comprising:
forming on a substrate surface a test body of liquid, wherein a
contact angle between the test body of liquid and the substrate
surface is lower than an equilibrium contact angle; analysing an
optical image of the test body of liquid to determine whether one
and only one cell is present in the test body of liquid.
2. The method of claim 1, wherein the contact angle between the
test body of liquid and the substrate surface is nearer to zero
than to the equilibrium contact angle.
3. The method of claim 1, wherein the forming of the test body of
liquid comprises: depositing a precursor body of liquid on the
substrate surface; and removing a portion of the precursor body of
liquid while the precursor body of liquid is in contact with the
substrate surface.
4. The method of claim 3, wherein the one and only one cell, where
present, originates from the precursor body of liquid.
5. The method of claim 3, wherein: the method further comprises
adding a further volume of liquid to an intermediate body of liquid
formed by the removing of the portion of the precursor body of
liquid, thereby providing the test body of liquid; and the one and
only one single cell, where present, is provided in the further
volume of liquid.
6. The method of claim 5, wherein the volume of the test body of
liquid, after the further volume of liquid has been added, is
smaller than the volume of the precursor body of liquid.
7. The method of claim 3, wherein the removing of the portion of
the precursor body of liquid comprises removing at least 50% of the
volume of the precursor body of liquid.
8. The method of claim 1, wherein the forming of the test body of
liquid comprises bringing a wetted body into contact with the
substrate surface and, subsequently, removing the wetting body from
contact with the substrate surface.
9. The method of claim 1, wherein the forming of the test body of
liquid comprises ejecting liquid from an ejection head while moving
the ejection head relative to the substrate surface in such a way
that a body of liquid is formed having a contact angle that is
lower than the equilibrium contact angle.
10. The method of claim 1, further comprising overlaying the test
body of liquid with an overlay liquid and wherein the analysed
optical image of the test body comprises an optical image of the
test body with the overlay liquid overlaying the test body of
liquid, the overlay liquid being immiscible with the test body of
liquid.
11. The method of claim 10, wherein the refractive index of the
overlay liquid is more similar to the refractive index of the test
body of liquid than to the refractive index of air.
12. The method of claim 1, wherein: the substrate surface forms at
least a portion of a boundary of a reservoir volume for cell
culturing; and the reservoir volume is at least partially filled
with liquid for cell culturing after it has been determined that
the test body of liquid contains one and only one cell.
13. The method of claim 12, further comprising culturing a
monoclonal colony of cells in the reservoir volume.
14. The method of claim 12 or 13, wherein the steps of forming and
analysing are repeated for a plurality of reservoir volumes and a
monoclonal colony of cells is cultured in each of the reservoir
volumes in which it is determined that the test body of liquid
contains one and only one cell.
15. The method of claim 14, wherein the plurality of reservoir
volumes are separated from each other by solid walls.
16. The method of claim 15, wherein each test body of liquid is
separated from all solid walls separating the reservoir volume from
other reservoir volumes.
17. The method of claim 14, wherein the plurality of reservoir
volumes are separated from each other by liquid walls.
18. A method of providing an isolated single cell, comprising:
providing a test body of liquid on a substrate surface, the test
body of liquid containing a single cell; overlaying the test body
of liquid with an overlay liquid immiscible with the test body of
liquid; and analysing an optical image of the test body of liquid
overlaid with the overlay liquid to determine whether the test body
of liquid comprises one and only one cell.
19. (canceled)
20. The method of claim 1, further comprising capturing the optical
image of the test body of liquid.
21. An apparatus for providing an isolated single cell, comprising:
a dispensing unit configured to form a test body of liquid on a
substrate surface in such a way that a contact angle between the
test body of liquid and the substrate surface is lower than an
equilibrium contact angle; an optical system configured to form an
optical image of the test body of liquid; and an analysis unit
configured to analyse the captured image to determine whether one
and only one cell is present in the test body of liquid.
22. (canceled)
Description
[0001] The invention relates to methods and apparatus for providing
isolated single cells, for example for monoclonal cell
culturing.
[0002] A wide range of applications involving monoclonal cell
cultures require that colonies of cells are produced that are known
with high reliability to be derived from a single cell.
Applications include, for example, therapeutic monoclonal antibody
production, stem cell therapy and gene editing. Within a well-plate
this is challenging and time consuming, and very often not possible
due to the so called "edge-effect" in which the solid walls of the
traditional microtiter plate interfere with optical measurements
for detecting the presence of cells. Micro-plates (or
microtiter/-well plates) are widely used during liquid handling;
each plate is essentially an array of miniature test tubes. Plates
have an accepted standard size (127.76.times.85.48.times.14.22 mm);
those with 96, 384, and 1,536 wells/plate are commercially
available and have working volumes per well of .about.100-500,
.about.15-150 and .about.3-10 microliters, respectively.
[0003] In addition to the edge effect, traditional well-plates
often require spinning down prior to imaging and/or labelling of
cells with fluorescent marker before they can be detected. These
additional processing steps add complexity and/or lengthen
processing times.
[0004] An alternative approach is to deposit small drops containing
cells onto localised regions in wells of a well-plate, with the
drops being small enough that they do not touch boundary walls of
the wells. The above-mentioned edge-effects are thus avoided.
Individual drops can be imaged from above or below to determine
whether a cell is present. Usually, light is made to pass through
the drops and is then imaged. Curvature in the upper interface of
each drop can reduce the quality of the image around the edges of
the drop. Time is also required to allow cells to fall to the
bottom of the drop and allow reliable optical detection.
[0005] It is an object of the invention to provide improved methods
and apparatus for providing isolated single cells.
[0006] According to an aspect of the invention, there is provided a
method of providing an isolated single cell, comprising: forming on
a substrate surface a test body of liquid, wherein a contact angle
between the test body of liquid and the substrate surface is lower
than an equilibrium contact angle; analysing an optical image of
the test body of liquid to determine whether one and only one cell
is present in the test body of liquid.
[0007] Thus, a method is provided in which a cell (e.g. in a small
volume of liquid) is introduced into a test body of liquid that is
flattened relative to an equilibrium droplet shape. The lower
curvature allows cells located close to edges of the test body of
liquid to be recognized optically with improved confidence. The
lower height of the test body of liquid (relative to an equilibrium
droplet) reduces the time required for a cell to settle onto the
substrate surface, which allows a high quality optical image of the
cell to be obtained quickly. The approach makes it possible to
determine whether or not a body of liquid comprises one and only
one cell quickly and reliably.
[0008] In an embodiment, the forming of the test body of liquid
comprises: depositing a precursor body of liquid on the substrate
surface; and removing a portion of the precursor body of liquid
while the precursor body of liquid is in contact with the substrate
surface. It has been found that this approach allows test bodies to
be produced quickly and easily, as well as providing a high level
of control over the final shape of each test body, and high
reproducibility.
[0009] In an embodiment, the one and only one cell is provided in
(i.e. originates from) the precursor body of liquid (i.e. the cell
is present before the precursor body is flattened). This approach
minimizes the number of processing steps required.
[0010] In an embodiment, the method further comprises adding a
further volume of liquid to an intermediate body of liquid formed
by the removing of the portion of the precursor body of liquid. In
an embodiment, the one and only one cell is provided in the further
volume of liquid. This approach provides a high probability of a
cell being present in the test body of liquid by avoiding the risk
of the cell being removed during removal of liquid to form the
(flattened) test body. Fluid dynamic effects furthermore mean that
a cell present in the further volume of liquid is more likely to
settle in a position towards a centre of the test body of liquid,
when the further volume of liquid is added, than a cell that is
present in the test body of liquid because the cell was already
provided in a precursor body of liquid.
[0011] In an embodiment, the test body of liquid is overlaid with
an overlay liquid and the analysed optical image of the test body
comprises an optical image of the test body with the overlay liquid
overlaying the test body of liquid. The overlay liquid is
immiscible with the test body of liquid. The overlay liquid reduces
the size of the refractive index change at the curved boundary of
the test body of liquid, thereby facilitating accurate imaging of
the test body of liquid even in regions close to the edges of the
test body of liquid.
[0012] According to an aspect of the invention, there is provided a
method of providing an isolated single cell, comprising: providing
a test body of liquid on a substrate surface, the test body of
liquid containing a single cell; overlaying the test body of liquid
with an overlay liquid immiscible with the test body of liquid; and
analysing an optical image of the test body of liquid overlaid with
the overlay liquid to determine whether the test body of liquid
comprises one and only one cell.
[0013] According to an alternative aspect of the invention, there
is provided a method of providing an isolated single cell,
comprising: forming on a substrate surface a test body of liquid,
wherein a contact angle between the test body of liquid and the
substrate surface is lower than 25 degrees; and analysing an
optical image of the test body of liquid to determine whether one
and only one cell is present in the test body of liquid.
[0014] According to an aspect of the invention, there is provided
an apparatus for providing an isolated single cell, comprising: a
dispensing unit configured to form a test body of liquid on a
substrate surface in such a way that a contact angle between the
test body of liquid and the substrate surface is lower than an
equilibrium contact angle; an optical system configured to form an
optical image of the test body of liquid; and an analysis unit
configured to analyse the captured image to determine whether one
and only one cell is present in the test body of liquid.
[0015] According to an aspect of the invention, there is provided
an apparatus for providing an isolated single cell, comprising: a
dispensing unit configured to provide a test body of liquid on a
substrate surface, and to overlay the test body of liquid with an
overlay liquid immiscible with the test body of liquid; an optical
system configured to form an optical image of the test body of
liquid overlaid with the overlay liquid; and an analysis unit
configured to analyse the captured image to determine whether one
and only one cell is present in the test body of liquid.
[0016] Embodiments of the invention will now be described, by way
of example only, with reference to the accompanying drawings in
which corresponding reference symbols indicate corresponding parts,
and in which:
[0017] FIG. 1 is an optical image of a cell near to a solid wall of
a well plate.
[0018] FIG. 2 is an optical image of a cell near to a liquid wall
of a reservoir volume separated from an adjacent reservoir volume
by a liquid wall.
[0019] FIG. 3 is an optical image of a cell doublet near to a
liquid wall of a reservoir volume separated from an adjacent
reservoir volume by a liquid wall.
[0020] FIG. 4 is a side sectional view depicting a portion of a
well plate and use of a dispensing unit to deposit a body of liquid
onto a substrate surface in a well and use of an optical system to
form an image of the body of liquid.
[0021] FIG. 5 is an optical image of a body of liquid of the type
depicted in FIG. 4.
[0022] FIG. 6 is a side sectional view depicting a portion of a
well plate and use of a dispensing unit to overlay a test body of
liquid with an overlay liquid, and use of an optical system to form
an image of the overlaid test body of liquid.
[0023] FIG. 7 is an optical image of an overlaid test body of
liquid of the type depicted in FIG. 6.
[0024] FIG. 8 is a side sectional view depicting a portion of a
well plate and use of a liquid removal unit to remove liquid from a
precursor body of liquid to provide a test body of liquid.
[0025] FIG. 9 is an optical image of a test body of liquid of the
type depicted in FIG. 8, formed by removing 80% of liquid from the
body of liquid imaged in FIG. 5.
[0026] FIG. 10 is a side sectional view depicting a portion of a
well plate and use of a dispensing unit to overlay a test body of
liquid of the type depicted in FIG. 8 with an overlay liquid, and
use of an optical system to form an image of the overlaid test body
of liquid.
[0027] FIG. 11 is an optical image of an overlaid test body of
liquid of the type depicted in FIG. 10, formed by overlaying the
test body of liquid imaged in FIG. 9.
[0028] FIG. 12 is a side sectional view depicting a portion of a
well plate and adding of a further volume of liquid to an
intermediate body of liquid to introduce a cell to the intermediate
body of liquid and form a test body of liquid.
[0029] FIG. 13 is a side sectional view depicting a portion of a
well plate showing wells after at least partial filling with liquid
for cell culturing.
[0030] FIG. 14 is a side sectional view of an alternative
embodiment in which reservoir volumes are separated from each other
by liquid walls rather than solid walls.
[0031] FIGS. 15-17 depict a sequence of operations for forming a
test body using an wetted body (e.g. an impregnated porous
material).
[0032] FIG. 18 depicts forming a test body by ejecting liquid from
a moving ejection head.
[0033] FIG. 19 depicts: (a) Sessile drop nomenclature. (b)
Illustration of light path passing through a sessile drop on a
polystyrene substrate. Different angles to the drop surface, a,
result in different exit angles .mu.. (c) The refracted light
enters the objective when .mu.<.mu..sub.m, and when
.mu.>.mu..sub.m dark regions appear on the image.
[0034] FIG. 20 depicts: Images (a) & (d-h) taken with 10.times.
objective with NA 0.25 (Olympus A10 PL) and image (i) taken with a
20.times. objective with NA 0.75 (Nikon Plan Apo) on IX53 inverted
microscope. (b) taken with FTA instrumentation. Base diameter is
1.68 mm for all drop images and the volume of each drop is
indicated. (a) Sessile drop on inverted microscope, (b) side view
of drop in (a), (c) plot of light intensity along indicated dotted
line in (a). (d-i) drop images with varying volume. Less volume
results in reduced curvature thereby reducing the maximum .mu. and
removing dark regions close to the pinning line visible.
[0035] FIG. 21 depicts: Identifying cells in well plates. All drops
have the same footprint area, with varying volumes indicated, and
image taken with a 10.times. objective with NA 0.25 (Olympus A10
PL). a(i)-d(i) Illustrations of the experimental setup in each
column. a(ii)-d(ii) Images of drops made with DMEM+10% FBS, c(ii)
& d(ii) drops submerged in FC40. a(iii)-d(iii) Same drop shape
as previous row with HEK cells in media prior to forming drops.
a(iv)-d(iv) Magnification of a portion of a(iii)-d(iii).
[0036] As discussed in the introductory part of the description,
edge-effects can interfere with reliable determination of whether a
single cell is present in a well of a well plate. The problem is
illustrated in the optical image of FIG. 1, where the presence of a
wall optically obscures a cell adjacent to the wall. In this
example, the cell is only identifiable by using expensive optics
and fluorescence or other labelling of the cell. Even with
expensive optics and labelling, the presence of the wall makes cell
identification less reliable and potentially more time consuming.
The magnitude of the edge-effect can be appreciated by comparing
the image of FIG. 1 with the images of FIGS. 2 and 3, which
respectively show how a single cell and a cell doublet can be
identified more easily when the solid wall is replaced by a liquid
wall.
[0037] Embodiments of the present disclosure provide methods and
apparatus which allow a single cell (i.e. one and only one cell) to
be verifiably introduced to a reservoir for monoclonal cell
culturing, or other methods requiring single cell isolation, with
improved reliability, speed and/or without requiring excessively
expensive equipment.
[0038] According to a class of embodiments, examples of which are
described in detail below with reference to FIG. 4 onwards, a
method of providing an isolated single cell comprises forming on a
substrate surface 4 a test body 12 of liquid, wherein a contact
angle between the test body 12 of liquid and the substrate surface
4 is lower than an equilibrium contact angle, optionally lower than
80%, optionally lower than 60%, optionally lower than 40%,
optionally lower than 20%, of the equilibrium contact angle. In an
embodiment, the contact angle between the test body 12 of liquid
and the substrate surface 4 is nearer to zero, optionally nearer to
the receding contact angle, than to the equilibrium contact angle.
The method further comprises analysing an optical image of the test
body 12 of liquid to determine whether one and only one cell is
present in the test body 12 of liquid. The method may comprise
capturing an optical image of the test body 12 of liquid and
analysing the captured image to determine whether one and only one
cell is present in the test body 12 of liquid.
[0039] The concept of a contact angle is well known in the art. The
contact angle is the angle where a liquid interface meets a solid
surface and quantifies the wettability of the solid surface for the
liquid in question. For a given system of solid, liquid and
vapour/liquid, there is a unique equilibrium contact angle. Contact
angle hysteresis is observed in practice, which means that contact
angles between a maximal (advancing) contact angle and a minimal
(receding) contact angle can be observed in certain circumstances.
Various methods are available for measuring contact angles,
including for example the static sessile drop method, the dynamic
sessile drop method, the single-fiber meniscus method, and the
Washburn's equation capillary rise method.
[0040] In an embodiment, as depicted in FIG. 4, a dispensing unit 2
is used to deposit liquid onto the substrate surface 4 in order to
provide the test body 12 of liquid. In an embodiment, as described
below, the dispensing unit 2 initially deposits a precursor body 11
of liquid, which is processed in subsequent steps to provide the
test body 12 of liquid. In an embodiment, the test body 12 and/or
precursor body 11 of liquid form a circular drop on the substrate
surface 4. In an embodiment, the substrate surface 4 forms a
boundary of a reservoir volume 6 for cell culturing. In this
example, the substrate surface 4 is the bottom surface of a well of
a well plate 8, each well of the well plate 8 providing a different
one of the reservoir volumes 6. The well plate 8 may take any of
the forms known in the art of well plates, including for example a
commercially available well plate. Non-limiting examples of well
plates that could be used include well plates having 96, 384, or
1,536 wells/plate, which may have working volumes per well of
.about.100-500, .about.15-150 and .about.3-10 microliters,
respectively. In the example of FIG. 4, only a small portion of the
well plate 8 is shown. In the interests of clarity, use of the
dispensing unit 2 is depicted for one of the wells only, but it
will be understood that the process can be repeated or performed in
parallel for multiple wells.
[0041] The nature of the dispensing unit 2 is not particularly
limited. Any dispensing unit 2 that is capable of depositing liquid
bodies with the required spatial and volumetric precision may be
used. The dispensing unit 2 may thus comprise any suitable
combination of liquid handling apparatus for this purpose,
including for example a suitably configured gantry system for
moving an injection head over the surface of the well plate 8 to
position the injection head over each well (e.g. piezo, inkjet
printer, pump and tubing) and a controller for directing injection
of a controlled amount of liquid onto a localized region within
each well. The dispensing unit 2 may comprise a plurality of
different devices and/or be configured to perform a plurality of
different techniques. The dispensing unit 2 may, for example, be
additionally configured to remove liquid and thereby act as a
liquid removal unit 18 (described below). The dispensing unit 2 may
be configured to add an overlay liquid 13 (described below). The
dispensing unit 2 may be configured to add a further volume 20 of
liquid containing a cell (described below). The dispensing unit 2
may be configured to add media to fill the reservoir, e.g. media
for cell culturing (described below).
[0042] In an embodiment, an optical system 14 (comprising, for
example, one or more lenses, an optical detector and/or a light
source) is provided for capturing an optical image of a body of
liquid (e.g. a test body 12 or a precursor body 11). The capturing
of the optical image may comprise viewing of the optical image by a
human and/or, where the capturing is at least partly performed by a
machine, storing data representing the optical image, at least
until the captured image is analysed (see below). The optical
system 14 may be configured such that a focal plane of the optical
image is coincident with, or near to, a plane of the substrate
surface 4. The optical system 14 may thus preferentially image a
portion of a body of liquid on the substrate surface 4 that is
directly adjacent to the substrate surface 4, thereby allowing
detection of a cell that has settled on the substrate surface 4
with high sensitivity. In an embodiment, the optical system 14 is
configured to provide illumination from above and image from below.
In an embodiment, an analysis unit 16 is provided that is
configured to analyse the captured image to determine whether a
single cell (i.e. one and only one cell) is present in the body of
liquid being imaged. Alternatively or additionally, the captured
image may be analysed (assessed) by a human operator, for example
while the optical image is being viewed by the operator using the
optical system 14 or while the operator is viewing a version of the
captured image displayed on a display, to determine whether a
single cell (i.e. one and only one cell) is present in the body of
liquid being imaged (or which has been imaged).
[0043] The analysis unit 16 may be computer-implemented. The
computer may comprise various combinations of computer hardware,
including for example CPUs, RAM, SSDs, motherboards, network
connections, firmware, software, and/or other elements known in the
art that allow the computer hardware to perform the required
computing operations. The required computing operations may be
defined by one or more computer programs. The one or more computer
programs may be provided in the form of media, optionally
non-transitory media, storing computer readable instructions. When
the computer readable instructions are read by the computer, the
computer performs the required method steps. The computer may
consist of a self-contained unit, such as a general-purpose desktop
computer, laptop, tablet, mobile telephone, smart device (e.g.
smart TV), etc. Alternatively, the computer may consist of a
distributed computing system having plural different computers
connected to each other via a network such as the internet or an
intranet.
[0044] In an embodiment, the analysis unit 16 uses a pattern
recognition algorithm to identify cells within the image captured
by the optical system 14. The analysis unit 16 determines that the
body of liquid contains one and only one cell when the pattern
recognition algorithm identifies one and only one cell in the
captured image.
[0045] In some embodiments, the optical system 14 images the body
of liquid from below. This ensures that the interface of the body
of liquid nearest to the optical system 14 is flat (if the
substrate surface 4 is flat), which helps produce a clear image. In
other embodiments, the optical system 14 images the body of liquid
from above.
[0046] FIG. 5 depicts an image of a body of liquid of the type
depicted in FIG. 4, consisting of a 1 .mu.l drop at equilibrium
(with an equilibrium contact angle between the liquid and the
substrate surface). Although the interface of the body of liquid
nearest to the optical system 14 is flat, the curvature of the
upper interface between the drop and air reduces the quality of the
image towards the edge of the body (the darker region near the
circumference of the circular drop) and makes it more difficult to
detect cells reliably in this region.
[0047] In an embodiment, as depicted schematically in FIG. 6, the
dispensing unit 2 overlays a test body 12 of liquid with an overlay
liquid 13. The test body 12 of liquid may in this case be formed by
overlaying a body of liquid that initially had an equilibrium
contact angle (or greater), such as the body of liquid illustrated
in FIG. 4. Alternatively, as described below, the test body 12 may
comprise a flattened body of liquid having a contact angle with
respect to the substrate surface 4 that is less than the
equilibrium contact angle. The overlay liquid 13 is immiscible with
the test body 12 of liquid. In an embodiment, the test body 12 of
liquid is aqueous and the overlay liquid 13 is immiscible with
water. In an embodiment the overlay liquid 13 comprises an oil. In
an embodiment, the overlay liquid 13 comprises a fluorocarbon such
as FC40, which is a transparent fully fluorinated liquid of density
1.8555 g/ml that is widely used in droplet-based microfluidics.
[0048] In an embodiment, the refractive index of the overlay liquid
13 is more similar to the refractive index of the test body 12 of
liquid (e.g. more similar to the refractive index of water) than to
the refractive index of air. This reduces the size of the
difference in refractive index at the curved upper boundary of the
test body 12 of liquid and, as shown in FIG. 7, thereby mitigates
the reduction in image quality towards the edge of the image of the
test body 12 and facilitates detection of cells in this region. The
improvement can be appreciated by comparing FIG. 5 with FIG. 7.
[0049] In an embodiment, as depicted in FIG. 8, the forming of the
test body 12 of liquid comprises depositing a precursor body 11 of
liquid (e.g. such as a body of liquid with a contact angle equal to
or greater than an equilibrium contact angle, such as the body of
liquid depicted in FIG. 4), and a liquid removal unit 18 is used to
remove a portion of the precursor body 11 of liquid while the
precursor body 11 of liquid is in contact with the substrate
surface 4. In an embodiment, at least 50% of the precursor body 11
of liquid is removed, optionally at least 60%, optionally at least
70%, optionally at least 80%, optionally at least 90%, optionally
at least 95%, optionally at least 99%. The removal is performed
such that a contact angle between the resulting body of liquid and
the substrate surface 4 is lower than a contact angle between the
precursor body 11 of liquid and the substrate surface 4. Thus, for
example, the precursor body 11 of liquid may be deposited onto the
substrate surface 4 in such a way that the contact angle between
the precursor body 11 of liquid and the substrate surface 4 is at
or near to an equilibrium contact angle. The removal of liquid may
then be implemented by sucking liquid out of the precursor body 11
so that the body of liquid becomes flatter. The contact angle is
thus reduced, for example to a contact angle that is between the
equilibrium contact angle and a receding contact angle or
approximately equal to the receding contact angle. The body of
liquid formed by the removal of liquid may be the test body 12 of
liquid, ready for imaging to determine whether one and only one
cell is present (as depicted in FIG. 8), or may, as described in
further detail below, be an intermediate body 121 of liquid to
which a further volume of liquid is added at a later stage to
supply a cell. Thus, the test body 12 may be a body that is flatter
than a precursor body 11 body but less flat than an intermediate
body 121. The composition of the liquid of the test body 12 (and,
where provided, the intermediate body 121) will normally be
substantially the same as the composition of the liquid of the
precursor body 11 (e.g. aqueous in both, or all, cases).
[0050] The nature of the liquid removal unit 18 is not particularly
limited. Any liquid removal unit 18 that is capable of removing
liquid with suitable accuracy may be used. The liquid removal unit
18 may thus comprise any suitable combination of liquid handling
apparatus for this purpose, including for example a suitably
configured gantry system for moving a suction head over the surface
of the well plate 8 to position the suction head over each well and
a controller for directing suction of a controlled amount of liquid
from a localized region within each well. In the interests of
clarity, use of the liquid removal unit 18 is depicted for one of
the wells only, but it will be understood that the process can be
repeated or performed in parallel for multiple wells.
[0051] In embodiments of this type, the optical system 14 captures
an image of a relatively flat test body 12 of liquid rather than of
a test body 12 that is near an equilibrium shape (e.g. as depicted
in FIG. 6) but may be otherwise configured as described above. The
captured image of the test body 12 of liquid is analysed, for
example by the analysis unit 16, to determine whether one and only
one cell is present in the test body 12 of liquid. Apart from the
fact that the image is derived from a flattened test body 12, the
analysis unit 16 may be otherwise configured as described
above.
[0052] FIG. 9 shows an optical image of a test body 12 of liquid of
the type depicted in FIG. 8, formed by removing 0.8 nl of liquid
from the body of liquid imaged in FIG. 5. The flattening caused by
the removal of liquid to form the test body 12 of liquid reduces
the curvature of the upper interface and mitigates the reduction in
image quality towards the edge of the image of the body of liquid
and facilitates detection of cells in this region. The improvement
can be appreciated by comparing FIGS. 5 and 9.
[0053] In an embodiment, as depicted schematically in FIG. 10, the
dispensing unit 2 overlays the flattened test body 12 of liquid
with an overlay liquid 13. The overlay liquid 13 may take any of
the forms described above with reference to FIGS. 6 and 7. The
overlay liquid 13 reduces the size of the difference in refractive
index at the curved upper boundary of the test body 12 of liquid
and, as shown in FIG. 11, thereby mitigates the reduction in image
quality towards the edge of the image of the test body 12 and
facilitates detection of cells in this region. The improvement can
be appreciated by comparing FIG. 5 or 9 with FIG. 11. Indeed, in
FIG. 11 the outer edge of the test body 12 is almost invisible.
[0054] In an embodiment, the one and only one cell, where present,
is provided in (i.e. originates from) the precursor body 11 of
liquid (where a precursor body of liquid 11 is used). As described
below, the precursor body 11 of liquid may initially be provided
with multiple cells but cells may be removed during the formation
of the test body 12. In embodiments where the one and only one cell
originates from the precursor body 11, no additional steps are
required to add cells. For example, cells may be provided in a
liquid used to deposit multiple precursor bodies 11 of the liquid,
with a concentration of the cells being such that a suitable number
of the precursor bodies 11 of liquid will, on average, contain one
and only one cell and/or that a suitable number of the test bodies
12 of liquid will contain one and only one cell (even after liquid
has been removed to form the test bodies 12 from the precursor
bodies 11). Thus, in some embodiments, particularly where a large
proportion of the precursor body 11 of liquid is removed to provide
a test body 12 of liquid, the precursor body 11 of liquid may
initially contain many cells, but with the concentration of the
cells in the precursor body 11 being such that when the test body
12 is formed there is a relatively high probability that the test
body 12 will contain one and only one cell.
[0055] Alternatively or additionally, as depicted in FIG. 12, the
dispensing unit 2 may be configured to add a further volume 20 of
liquid to an intermediate body 121 of liquid, the intermediate body
121 of liquid being a body of liquid formed by removing a portion
of a precursor body 11 of liquid (e.g. as described above with
reference to FIG. 8). The body of liquid resulting from the
addition of the further volume 20 of liquid to the intermediate
body 121 of liquid is the test body 12 of liquid ready for imaging
and determination of whether one and only one cell is present in
the test body 12. The one and only one cell, where present, is
provided in the further volume 20 of liquid. The further volume 20
of liquid may be added using single-cell printer technology, for
example. In an embodiment, cells are imaged in an ejection head to
identify when a single isolated cell is present in a volume of
liquid (near a tip) to be ejected and, when a single cell is
identified by the imaging, the volume of liquid to be ejected is
ejected as the further volume 20 of liquid. Thus, a cell may be
added after an intermediate body 121 of liquid has been formed by
removing liquid from a precursor body 11 of liquid. This approach
may facilitate localisation of the cell towards the centre of the
reservoir volume due to fluid dynamic effects, which will favour
coalescence of the further volume 20 with the intermediate body 121
in such a way that any cell in the further volume 20 will tend to
be localised more towards the centre of the resulting test body 12
than towards the edges of the resulting test body 12. Liquid in the
further volume 20 is typically added to the intermediate body 121
near the centre which causes liquid already in the intermediate
body 121 to be displaced outwards whereas the newly added liquid
remains near the centre.
[0056] In an embodiment, the further volume 20 is small enough that
the test body 12 of liquid remains relatively flat even though the
test body 12 has been formed by addition of the further volume 20
to the intermediate body 121, thereby ensuring that the curvature
of the upper interface of the test body 12 remains relatively low
and allows reliable detection of a single cell in the test body 12
by the optical system 14. In an embodiment, the volume of the test
body 12 of liquid, after the further volume 20 of liquid has been
added, is smaller than the volume of the precursor body 11 of
liquid. In the example described above in which a precursor body 11
having a volume of approximately 1 .mu.l is provided (FIG. 4) and
800 nl is removed to form the intermediate body 121, the further
volume 20 will thus be less than 800 nl. In an embodiment, the
further volume 20 is applied using a single cell printer method,
such as a drop generating nozzle.
[0057] In the embodiments described above, methods are described in
which a flatter than equilibrium body of liquid (e.g. the test body
12 or the intermediate body 121) is formed by removing liquid from
a precursor body 11. In other embodiments, a flatter than
equilibrium body of liquid (suitable for acting as a test body 12
or an intermediate body 121) is formed by directly depositing the
body of liquid in the flattened form. In one class of embodiments,
as depicted in FIGS. 15-17, this is achieved by bringing a wetted
body 26 (e.g. a porous material impregnated with liquid, such as a
humid sponge, or a solid member having a body of water formed on
it) into contact with the substrate surface 4 continuously over a
contact region (which may be referred to as a wetted region) on the
substrate surface 4 and then removing the wetted body 26. This
approach could directly provide a body of liquid spanning the
contact region with a contact angle that is lower than the
equilibrium contact angle. In another class of embodiments, as
depicted in FIG. 18, a forward printing process may be performed in
which liquid is ejected onto the substrate surface 4 from an
ejection head 28 while the ejection head 28 is moved relative to
the substrate surface 4 in such a way that a body of liquid is
formed having a contact angle that is lower than an equilibrium
contact angle. This can be achieved by suitable control of the rate
of flow of liquid out of the ejection head 28 and the speed of
movement of the ejection head 28 relative to the substrate surface
4 (e.g. so the rate of flow is not too high and the speed of
movement is not too low). In yet another class of embodiments, a
test body 12 is formed that has a very low equilibrium contact
angle, optionally lower than 25 degrees (in air and/or when
overlaid with an overlay liquid 13 such as FC40), optionally lower
than 15 degrees, optionally lower than 10 degrees, optionally lower
than 5 degrees, optionally lower than 1 degree. Thus, the benefits
related to having a relatively flat test body 12 discussed above
can be achieved without necessarily using steps to achieve a
contact angle that is less than the equilibrium contact angle
(although such steps may be employed to further reduce the contact
angle). Various techniques are known for achieving low equilibrium
contact angles, including adding surfactants to the liquid. In an
exemplary embodiment, a test body 12 is formed that contains a
poloxamer such as a Pluronic.RTM., which is known to be
particularly compatible with cells. Poloxamers are nonionic
triblock copolymers composed of a central hydrophobic chain of
polyoxypropylene flanked by two hydrophilic chains of
polyoxyethylene. In another exemplary embodiment, a test body 12 is
formed which contains Polysorbate 20, which is a polysorbate-type
nonionic surfactant formed by the ethoxylation of sorbitan before
the addition of lauric acid. Many other non-ionic surfactants could
be used with low risk of damage to cells as long as the
concentration/exposure time is kept sufficiently low.
[0058] In an embodiment, each reservoir volume 6 is at least
partially filled with liquid for cell culturing after it has been
determined that the test body 12 of liquid comprises one and only
one cell, as depicted in FIG. 13. The at least partial filling may
be such that all of a base of each reservoir volume 6 is entirely
covered by liquid. In an embodiment, each reservoir volume 6 is
filled up to at least 25% (optionally at least 50%, optionally at
least 75%) of the height of the reservoir volume 6. In an
embodiment, a process of culturing a monoclonal colony of cells is
then performed in each reservoir volume 6 for which it has been
determined that one and only one cell is initially present. The
process of culturing may comprise ensuring that the cells have
access to any nutrients, growth factors, hormones and/or gases that
may be needed, as well as controlling the physio-chemical
environment to maintain suitable conditions. The at least partial
filling of each reservoir volume 6 with the liquid for cell
culturing may be performed starting from any of the configurations
depicted in FIGS. 6, 8 and 10. Where an overlay liquid 13 is
provided, the overlay liquid 13 may be removed or partially removed
prior to the filling with the liquid for cell culturing or the
overlay liquid 13 may be left and removed at a later stage (or not
removed at all).
[0059] The processes described above (e.g. the forming of the test
body 12 of liquid, the optional removal of liquid to provide the
test body 12, the optional overlaying, the imaging and analysis
steps) are repeated for a plurality of reservoir volumes 6 (e.g.
all of the reservoir volumes 6 defined by respective wells in a
well plate) and a monoclonal colony of cells is cultured in each of
the reservoir volumes 6 in which it is determined that one and only
one cell is initially present. In an embodiment, the plurality of
reservoir volumes 6 are separated from each other by solid walls 22
(as depicted in FIG. 13). In an embodiment, each test body 12 of
liquid is provided in a central region of a respective reservoir
volume 6 so as not to be in contact with any of the solid walls 22
separating the reservoir volume 6 from other reservoir volumes
6.
[0060] In an alternative embodiment, the plurality of reservoir
volumes 6 are separated from each other by liquid walls 24 (as
depicted in FIG. 14). The reservoir volumes 6 in this case may be
formed by adding liquid for cell culturing to the test bodies 12 of
liquid after detection of single cells has been performed. The
added liquid may be such that a footprint of each reservoir volume
6 on the substrate surface 4 is the same as the footprint of the
respective corresponding test body 12 of liquid (by ensuring that
contact angle of each reservoir volume 6 with the substrate surface
4 does not exceed the advancing contact angle). The plurality of
reservoir volumes 6 are overlaid with an overlay liquid 13. The
overlay liquid may take any of the forms discussed above (e.g.
FC40). The liquid walls 24 are thus formed from the overlay liquid
13 between the reservoir volumes 6.
Background Theory and Further Experimental Validation
[0061] References in the discussion below to "drops" should be
understood to encompass bodies (e.g. test bodies) of liquid formed
on a substrate surface, as described above.
Theory
[0062] The maximum angle, .mu..sub.m, for which light rays are
accepted by a microscope objective in air can be calculated with
knowledge of the numerical aperture (NA)
.mu..sub.m=sin.sup.-1 NA
[0063] Light rays with angles that exceed .mu..sub.m will not reach
the image plane and hence may result in dark regions. As light rays
pass through a curved liquid surface, such as drop (e.g. a test
body of liquid on a substrate surface), the change in refractive
index results in the light being refracted in accordance with
Snell's law. To illustrate this effect a water sessile drop, with
refractive index n=1.33, is placed on a polystyrene, n=1.58,
surface (common well plate material) surrounded by air, n=1. If the
drop radius is less than the capillary length
( .lamda. c < .gamma. .DELTA. .rho. g ) ##EQU00001##
then gravity has a negligible effect and the drop has the shape of
a spherical cap. For a spherical cap, depicted in FIG. 19(a), the
maximum height, h, radius of curvature, R, volume, V.sub.cap,
footprint radius, a, and contact angle, .theta. are related
through
V c a p = .pi. ( 2 - 3 cos .theta. + cos 3 .theta. ) 3 a 3 sin 3
.theta. h = a sin .theta. ( 1 - cos .theta. ) R = h 2 + a 2 2 h
##EQU00002##
For a known volume and footprint radius the entire drop geometry
can be evaluated. Then, with reference to FIG. 19(a & b), the
angle between a tangent at any point on the surface and the
horizontal, a, is provided by
.alpha. = sin - 1 R a R ##EQU00003##
Using this angle a light ray trajectory through a sessile drop can
be determined by satisfying Snell's law of refraction. FIG. 19(c)
illustrates the path of parallel light through a sessile drop on a
polystyrene substrate with an air/water interface (solid light ray
paths) and FC40/water interface (broken light ray paths). If the
light rays are refracted such that their exit angle, .mu., exceeds
.mu..sub.m; then the image will appear dark in those regions as
shown in FIG. 19(c), where the indicated R.sub.a coincides with
.mu.=.mu..sub.m, white regions .mu.<.mu..sub.m and dark regions
.mu.>.mu..sub.m. When the drop is overlaid with an immiscible
fluid such as a fluorocarbon, FC40 with n=1.29, .mu. is lower than
air due to the higher refractive index of the immiscible fluid as
illustrated by the broken line light ray paths.
Experimental Setup
[0064] To identify single cells in wells plates it is important
that the entire region where cells may be deposited in a well have
optical clarity; in general, enhanced optical clarity lowers the
microscopy and labour/time costs. The principle of replacing the
solid wall, with fluid ones--the liquid/fluid interface of the drop
becomes the bounding fluid wall--and controlling volumes therein
enables complete clarity over the entire drop region with low cost
microscope objectives. This method was validated through placing
eight 1 .mu.l sessile drops of cell media (DMEM+10% Fetal Bovine
Serum (FBS)) with equal volume on a polystyrene substrate. Fluid
was extracted from seven, leaving drops with volumes between
100-1000 nl with constant footprint area--the FBS prevents the
pinning line from receding as fluid is removed from the drop as it
results in a low receding contact angle. Drops were imaged less
than ten seconds after being formed to minimise evaporation effects
using a Nikon D610 DSLR mounted on an IX53 inverted microscope,
operating in bright field mode, fitted with a 10.times.
objective--Olympus A10 PL; NA=0.25. The method works in both bright
field and phase contrast microscopy, although the former was
exclusively used herein. Contact angles, .theta., were calculated
as described in the theory section using the measured footprint
area (from images using a microscope calibration ruler); and also
measured directly by the sessile drop method using First Ten
Angstroms (FTA) instrument and software. For the latter method
drops were formed by ejecting a 1 .mu.l drop using a needle (33G
blunt NanoFil.TM. needle, World Precision Instruments) connected to
a syringe pump (Harvard Ultra) through a Teflon tube. The drops
were gently transferred to the surface of a square cut from the
base of a Corning.RTM. 60 mm suspension culture dish made from
polystyrene, and then imaged from the side. The resultant
equilibrium contact angles in air were found to be
.about.82.degree. and -80.degree. using the analytical and sessile
drop methods, respectively. Cells were prepared as previously
described.
Results
[0065] The drop images of FIG. 20 were processed to measure the
radius, R.sub.a, where the dark region begins, from which a and
light ray paths can be calculated; the resultant data is shown in
Table 1 for a range of drop volumes with constant footprint
diameter. Considering drops A-F (labelled a-f in FIG. 20); a ray of
light entering the drop vertically at R.sub.a gives an average
.mu.=12.8.degree. (SD of 0.6.degree.). The NA of the utilised
objective provides .mu..sub.m=14.5.degree.. Considering the
simplified assumption of parallel light entering the drop, this
agreement is satisfactory; moreover the approximately constant
value of .mu. for a range of drop volumes further illustrates the
validity of the analysis. The maximum refracted angle for all drops
considered occurs at the edge of the identical drops A & I (a
and i in FIG. 20) as .mu.=48.degree.; this is the largest value of
.alpha.=.theta.=82. A microscope objective with NA=0.75, giving
.mu..sub.m=48.6.degree., was used to view a portion of this drop
and the dark regions in the resultant image were minimized as shown
in FIG. 20(i). Table 1 also shows that drops G-H result in
.mu.<10.degree. everywhere and hence dark regions are eliminated
as shown in FIG. 20(g&h) using the NA=0.25 objective. The value
of .mu. can be reduced further by overlaying the drops with an
immiscible fluid of refractive index greater than air such as FC40
(a fluorocarbon with n=1.29); .mu. reduces from an averages of
12.8.degree. in air to 1.3.degree. for drop geometries A-F in Table
1.
TABLE-US-00001 TABLE 1 Volume R.sub.a h Drop (.mu.l) (.mu.m)
.theta..degree. (.mu.m) .alpha..degree.@R.sub.a
.mu..degree.@.alpha.(air) .mu..degree.@.alpha.(FC40) A 1 466 82 726
33.6 12.0 1.5 B 0.8 485 73 618 33.8 12.1 1.1 C 0.7 531 68 558 36.0
13.0 1.2 D 0.6 581 61 492 37.5 13.7 1.3 E 0.4 696 45 346 36.1 13.1
1.2 F 0.3* 835 35 263 35.0 12.6 1.2 G 0.2* 835 24 177 24.0 8.2 0.8
H 0.1* 835 13 91 12.5 4.2 0.4 I 1** 835 82 726 82 48 10.9 *Data
shown for completeness only and calculation based on .alpha. at the
pinning line. **Calculations for maximum refraction at maximum
contact angle of all drops considered.
Table 1: Geometric parameter calculated for the drop images shown
in FIG. 20 (some not shown) assuming drop shape is represented by
the cap of a sphere.
[0066] To evaluate the ease with which cells can be identified
using this method, four drops were placed on a suspension cell
culture substrate with varying volumes, and constant footprint, as
illustrated in 21a(i)-d(i). The drops were imaged as before and the
influence of the FC40/water interface is evident through comparison
between a(ii) and d(ii), where the dark annular region almost
disappears with FC40 overlay. This is also evident between b(ii)
and c(ii) where the outline of the drop almost disappears when
overlaid with FC40 and hence provides perfect clarity for
identifying cells in those regions. Drops created with cell
suspension are shown in a-d(iii), and a section digitally magnified
in a region near the pinning line in a-d(iv) to illustrate the ease
of identify cells.
[0067] The cells in a(iv) are impossible to see in the dark
regions, but single cell identification is possible in b-d(iv) with
low cost microscopy. It is noted that with the d(iv) method the
drop can still have substantial height, see Table 1 for numerical
values, and hence cell may be outside of the microscope objective
focal depth; two such cells are indicated in the image where there
outline is visible but they are out of the focus. Hence, this
approach would either require a vertical scan of the drop, or a
settling period for the cells to fall to the base of the dish
before commencing imaging to assure monoclonality. A high NA
objective lens, as used in 20(i) would also remove the dark regions
for the drops in column a, however higher costs, settling time
issues as in d(iv), and higher magnification (higher NA lens
typically have higher magnification or require specialised
microscope) would make its use of limited benefit.
[0068] The approach of b(iv) & c(iv), where the drop height is
reduced can remove the need for multiple images through the drop,
and settling time, by forcing the drop to be sufficiently flat.
Both of these approaches are efficient methods for identifying
cells within well plate formats and appear good approaches for
assuring monoclonality. A practical implementation of the method
for single cell isolation and assurance of monoclonality could be;
1) form media drop in a well plate to fit in single image, 2)
removing fluid from drop, 3) dispense nano-litres of single cell
suspension into the drop using established low volume dispensing
techniques, 3a) optionally overlay with FC40 if evaporation is
problematic, 4) record image and confirm which wells contain a
single cell, 5) fill the wells with media and process well plates
as normal.
[0069] Further embodiments of the disclosure are defined in the
following numbered clauses:
[0070] 1. A method of providing an isolated single cell,
comprising: forming on a substrate surface a test body of liquid,
wherein a contact angle between the test body of liquid and the
substrate surface is lower than an equilibrium contact angle;
capturing an optical image of the test body of liquid; and
analysing the captured image to determine whether one and only one
cell is present in the test body of liquid.
[0071] 2. The method of clause 1, wherein the contact angle between
the test body of liquid and the substrate surface is nearer to a
receding contact angle than to the equilibrium contact angle.
[0072] 3. The method of clause 1 or 2, wherein the forming of the
test body of liquid comprises: depositing a precursor body of
liquid on the substrate surface; and removing a portion of the
precursor body of liquid while the precursor body of liquid is in
contact with the substrate surface.
[0073] 4. The method of clause 3, wherein the one and only one
cell, where present, is provided in the precursor body of
liquid.
[0074] 5. The method of clause 3, wherein: the method further
comprises adding a further volume of liquid to an intermediate body
of liquid formed by the removing of the portion of the precursor
body of liquid, thereby providing the test body of liquid, the
further volume of liquid being added before the capturing of the
optical image of the test body of liquid; and the one and only one
single cell, where present, is provided in the further volume of
liquid.
[0075] 6. The method of clause 5, wherein the volume of the test
body of liquid, after the further volume of liquid has been added,
is smaller than the volume of the precursor body of liquid.
[0076] 7. The method of any of clauses 3-6, wherein the removing of
the portion of the precursor body of liquid comprises removing at
least 50% of the volume of the precursor body of liquid.
[0077] 8. The method of any preceding clause, wherein the forming
of the test body of liquid comprises bringing a wetted body into
contact with the substrate surface and, subsequently, removing the
wetting body from contact with the substrate surface.
[0078] 9. The method of any preceding clause, wherein the forming
of the test body of liquid comprises ejecting liquid from an
ejection head while moving the ejection head relative to the
substrate surface in such a way that a body of liquid is formed
having a contact angle that is lower than the equilibrium contact
angle.
[0079] 10. The method of any preceding clause, further comprising
overlaying the test body of liquid with an overlay liquid before
the capturing of the optical image of the test body of liquid, the
overlay liquid being immiscible with the test body of liquid.
[0080] 11. The method of clause 10, wherein the refractive index of
the overlay liquid is more similar to the refractive index of the
test body of liquid than to the refractive index of air.
[0081] 12. The method of any preceding clause, wherein: the
substrate surface forms at least a portion of a boundary of a
reservoir volume for cell culturing; and the reservoir volume is at
least partially filled with liquid for cell culturing after it has
been determined that the test body of liquid contains one and only
one cell.
[0082] 13. The method of clause 10, further comprising culturing a
monoclonal colony of cells in the reservoir volume.
[0083] 14. The method of clause 12 or 13, wherein the steps of
forming, capturing and analysing are repeated for a plurality of
reservoir volumes and a monoclonal colony of cells is cultured in
each of the reservoir volumes in which it is determined that the
test body of liquid contains one and only one cell.
[0084] 15. The method of clause 14, wherein the plurality of
reservoir volumes are separated from each other by solid walls.
[0085] 16. The method of clause 15, wherein each test body of
liquid is separated from all solid walls separating the reservoir
volume from other reservoir volumes.
[0086] 17. The method of clause 14, wherein the plurality of
reservoir volumes are separated from each other by liquid
walls.
[0087] 18. A method of providing an isolated single cell,
comprising: providing a test body of liquid on a substrate surface,
the test body of liquid containing a single cell; overlaying the
test body of liquid with an overlay liquid immiscible with the test
body of liquid; capturing an optical image of the test body of
liquid overlaid with the overlay liquid; and analysing the optical
image to determine whether the test body of liquid comprises one
and only one cell.
[0088] 19. A method of providing an isolated single cell,
comprising: forming on a substrate surface a test body of liquid,
wherein a contact angle between the test body of liquid and the
substrate surface is lower than 25 degrees; capturing an optical
image of the test body of liquid; and analysing the captured image
to determine whether one and only one cell is present in the test
body of liquid.
[0089] 20. An apparatus for providing an isolated single cell,
comprising: a dispensing unit configured to form a test body of
liquid on a substrate surface in such a way that a contact angle
between the test body of liquid and the substrate surface is lower
than an equilibrium contact angle; an optical system configured to
form an optical image of the test body of liquid; and an analysis
unit configured to analyse the captured image to determine whether
one and only one cell is present in the test body of liquid.
[0090] 21. An apparatus for providing an isolated single cell,
comprising: a dispensing unit configured to provide a test body of
liquid on a substrate surface, and to overlay the test body of
liquid with an overlay liquid immiscible with the test body of
liquid; an optical system configured to form an optical image of
the test body of liquid overlaid with the overlay liquid; and an
analysis unit configured to analyse the captured image to determine
whether one and only one cell is present in the test body of
liquid.
* * * * *